Expression of macrophage inflammatory protein-3alpha, stromal cell-derived factor-1, and B-cell-attracting chemokine-1 identifies the tonsil crypt as an attractive site for B cells.

The expression of 3 lymphoid chemokines-macrophage inflammatory protein-3alpha (MIP-3alpha), stromal cell-derived factor-1 (SDF-1), and B-cell-attracting chemokine-1 (BCA-1)-in the tonsil and the possible correlation between their sites of expression and B-cell localization within this tissue were studied. The results show that all 3 chemokines are produced in the crypts but differ by the nature of the cells that produce them and their location within the crypt. SDF-1 and MIP-3alpha are produced by epithelial cells, but their secretion is mutually exclusive. Both MIP-3alpha- and SDF-1-expressing cells are in close contact with memory B cells. By contrast, BCA-1-producing cells in the crypt are not epithelial and form clusters colocalized with plasma cells. Altogether, these data suggest that the chemokines produced in the tonsillar crypt may (1) attract memory B cells to antigen and (2) recruit and retain plasma cells and memory B cells within the supportive epithelial microenvironment of the crypt. (Blood. 2001;97:3992-3994)

Mitochondria connects the antigen receptor to effector caspases during B cell receptor-induced apoptosis in normal human B cells.

We have previously reported that CD40 stimulation sensitizes human memory B cells to undergo apoptosis upon subsequent B cell receptor (BCR) ligation. We have proposed that activation stimuli connect the BCR to an apoptotic pathway in mature B cells and that BCR-induced apoptosis of activated B cells could serve a similar function as activation-induced cell death in the mature T cell compartment. Although it has been reported that caspases are activated during this process, the early molecular events that link the Ag receptor to these apoptosis effectors are largely unknown. In this study, we report that acquisition of susceptibility to BCR-induced apoptosis requires entry of memory B cells into the S phase of the cell cycle. We also show that transduction of the death signal via the BCR sequentially proceeds through a caspase-independent and a caspase-dependent phase, which take place upstream and downstream of the mitochondria, respectively. Furthermore, our data indicate that the BCR-induced alterations of the mitochondrial functions are involved in activation of the caspase cascade. We have found both caspases-3 and -9, but not caspase-8, to be involved in the BCR apoptotic pathway, thus supporting the notion that initiation of the caspase cascade could be under the control of the caspase-9/Apaf-1/cytochrome c multimolecular complex. Altogether, our findings establish the mitochondria as the connection point through which the Ag receptor can trigger the executioners of apoptotic cell death in mature B lymphocytes.

Activation sensitizes human memory B cells to B-cell receptor-induced apoptosis.

The outcome of antigen receptor (B-cell receptor; BCR) ligation on B-cell survival can be influenced by multiple parameters. They are linked to the physical properties of the antigen itself, the maturational stage of the cells and the costimuli provided by different components of the innate and acquired immunity. Here we report that apoptosis prevails over stimulation when a BCR agonist is applied to human memory B cells which have been preactivated by CD40 ligand or anti-immunoglobulin antibodies. The susceptibility of activated memory B cells to BCR-induced killing is correlated with their enhanced expression of the transcripts encoding the pro-apoptotic molecules Bax, c-Myc and p53. The BCR-mediated apoptosis of activated memory B cells does not require extensive cross-linking of the antigen receptors and relies neither on engagement of the FcgammaRII nor on the Fas/Fas ligand (Fas-L) system. Our findings suggest that activation stimuli open the BCR-induced apoptotic pathway in memory B cells. Therefore we propose that the concept of activation-induced cell death (AICD), originally described for T cells, also applies to mature B lymphocytes. The functions fulfilled by the AICD of mature B cells in the regulation of B-cell responses are discussed.

Modulation and functional involvement of CB2 peripheral cannabinoid receptors during B-cell differentiation.

Two subtypes of G-protein-coupled cannabinoid receptors have been identified to date: the CB1 central receptor subtype, which is mainly expressed in the brain, and the CB2 peripheral receptor subtype, which appears particularly abundant in the immune system. We investigated the expression of CB2 receptors in leukocytes using anti-CB2 receptor immunopurified polyclonal antibodies. We showed that peripheral blood and tonsillar B cells were the leukocyte subsets expressing the highest amount of CB2 receptor proteins. Dual-color confocal microscopy performed on tonsillar tissues showed a marked expression of CB2 receptors in mantle zones of secondary follicles, whereas germinal centers (GC) were weakly stained, suggesting a modulation of this receptor during the differentiation stages from virgin B lymphocytes to memory B cells. Indeed, we showed a clear downregulation of CB2 receptor expression during B-cell differentiation both at transcript and protein levels. The lowest expression was observed in GC proliferating centroblasts. Furthermore, we investigated the effect of the cannabinoid agonist CP55,940 on the CD40-mediated proliferation of both virgin and GC B-cell subsets. We found that CP55,940 enhanced the proliferation of both subsets and that this enhancement was blocked by the CB2 receptor antagonist SR 144528 but not by the CB1 receptor antagonist SR 141716. Finally, we observed that CB2 receptors were dramatically upregulated in both B-cell subsets during the first 24 hours of CD40-mediated activation. These data strongly support an involvement of CB2 receptors during B-cell differentiation.

A role for the VLA-4 integrin in the activation of human memory B cells.

It is generally recognized that activation through membrane effector molecules such as CD40 or the B cell receptor (BCR) is mandatory to allow B cells to proliferate and differentiate into antibody (Ab)-secreting cells in response to cytokines. We show here that purified tonsillar B cells can be stimulated directly by a cytokine combination to proliferate and secrete immunoglobulins when cultures are performed at high cell density. The contact-mediated activation of B cells in this experimental system is strongly inhibited both by anti-very late antigen (VLA)-4 monoclonal Ab and by a peptide containing the LDV sequence specifically recognized by the alpha 4 integrin binding site. These reagents also significantly suppressed the B cell responses elicited by engagement of the BCR or CD40. Our data reveal that memory B cells but not virgin or germinal center B cells are sensitive to the direct stimulatory effect of cytokines in high-density cultures. Finally, we found that the dual expression of the alpha and beta chains of VLA-4 is a distinctive feature of the memory B cell population. Collectively, our findings support the notion that VLA-4-dependent homotypic B cell interactions can mediate a co-stimulatory signal to human memory B cells and might participate in the B cell activation triggered through the BCR and CD40.

Antigen receptor-induced apoptosis of human germinal center B cells is targeted to a centrocytic subset.

The outcome of the signals transduced through the B cell antigen receptor (BCR) depends both on their maturational stage and on the extent of receptor cross-linking. It is established that the BCR-mediated apoptosis of immature B cells represents an important mechanism for tolerance induction in the pre-immune B cell compartment. We show here that mature germinal center (GC) B cells can re-acquire sensitivity to BCR-induced cell death following CD40 ligation. In contrast, neither virgin nor memory B cells become susceptible to antigen receptor-triggered apoptosis upon CD40 stimulation, suggesting that this phenomenon may play a role in the shaping of the mature B cell repertoire in GC. Our data reveal that the death signal evoked through the BCR does not involve the Fcgamma receptors, does not operate through the Fas/Fas ligand system, and can be blocked by interleukin-4. Finally, we found that the acquisition of sensitivity to the death-promoting effect of anti-Ig antibodies in CD40-stimulated GC B cell cultures correlates with the induction of a centrocytic phenotype. We propose that negative regulatory signals via the BCR may delete somatically mutated centrocytes with self-reactivity.

Identification of a tonsil IgD+ B cell subset with phenotypical and functional characteristics of germinal center B cells.

We have identified and isolated a subpopulation of IgD+ B cells (IgD+CD38+ B cells) from human tonsils which expresses the germinal center (GC)-associated surface markers CD10, CD38, CD75, CD77 and Cd95/Fas. The heterogeneity of expression of several markers on IgD+ CD38+ B cells suggests that this population can be further subdivided into two discrete subtypes. On a functional basis, IgD+ CD38+ B cells behave as GC B cells as they rapidly and spontaneously undergo apoptosis in vitro and cannot be stimulated to synthesize DNA upon cross-linking of the antigen receptor. However, in contrast with most GC B cells, IgD+ CD38+ B cells have not completed Ig class switching since they predominantly secrete IgM following stimulation in vitro and lack surface expression of secondary isotypes. Immunoenzymatic staining performed on tonsil tissue sections revealed that IgD+ CD38+ B cells are located in two distinct histological structures: within the GC of a few classical secondary follicles, in which they appear as scattered cells, and within rare atypical GC, homogeneously constituted of IgD+ B cells. Taken together, our findings indicate that IgD+ CD38+ B cells constitute a novel subset of GC B cells. The possibility that these cells could represent an early stage of the follicular reaction or be generated in response to certain bacterial carbohydrate antigens is discussed.

Concurrent engagement of CD40 and the antigen receptor protects naive and memory human B cells from APO-1/Fas-mediated apoptosis.

Naive and memory B cells were isolated from human tonsils and examined for expression of APO-1/Fas and for their sensitivity to the APO-1-dependent apoptosis. APO-1 was found to be constitutively expressed on memory but not on naive B cells. The susceptibility of both cell types to the APO-1 apoptotic pathway was acquired upon CD40 triggering and was correlated with increased expression of the APO-1 receptor. Both naive and memory B cells were protected from the APO-1-mediated death signal after dual ligation of the Ag receptor adn CD40. Our findings suggest that the APO-1 pathway controls the specificity of B cell responses to T-dependent Ags and that occupancy of the Ag receptor dictates the outcome of APO-1-ligation on B cell survival.